Our long-term OBJECTIVES are to apply the fundamental concepts and technologies of cell and molecular biology to understand the role of cholangiocytes, the epithelial cells lining intrahepatic bile ducts, in health and disease. Here we concentrate on cholangiocyte recognition and response to pathogens or pathogen-derived products. The biliary tract is periodically exposed to microbial-derived toxins (e.g. lipopolysaccharide) or colonized by pathogenic microorganisms (e.g. Cryptosporidium parvum, a parasitic protozoon and Category B Biodefense pathogen that causes intestinal and biliary cryptosporidiosis). Recent evidence from our lab indicates that: (i) C. parvum infection or treatment of cultured cholangiocytes with the TLR4 agonist, LPS, promotes the rapid activation of the small GTPase, N-Ras; (ii) activated N-Ras contributes to cholangiocyte production of proinflammatory cytokines and pathogen-induced proliferation; (iii) C. parvum and LPS alter the expression of the cholangiocyte microRNA expression profile, including the let-7 family; (iv) manipulation of selected microRNA expression (i.e. let-7i) influences post-transcriptional regulation of targeted proinflammatory mRNAs in cholangiocytes, including N-Ras; and, (v) our in vivo models of biliary responses to microbial stimuli reflect our in vitro observations of increased proinflammatory responses. Thus, we will test the CENTRAL HYPOTHESIS that pathogen-induced TLR4-dependent Ras activation promotes the cholangiocyte innate immune phenotype (cytokine production, increased proliferation) through transcriptional suppression of the let- 7 microRNAs. In our three integrated SPECIFIC AIMS we will test the hypotheses that: (i) cholangiocyte pathogen recognition activates N-Ras through a TLR-dependent mechanism, and activated N-Ras initiates proinflammatory cytokine production; (ii) pathogen-induced N-Ras activation in cholangiocytes suppresses let- 7 microRNA transcription, which promotes an increase in let-7 proinflammatory target proteins; and, (iii) pathogen-induced N-Ras activation and reduction of let-7 promote cholangiocyte proinflammatory responses in vivo and are essential for inflammatory cell infiltration. This proposal incorporates new in vitro techniques and animal models, novel hypotheses regarding the role of proinflammatory signaling axes that regulate and are regulated by microRNAs, and gene regulation by microRNAs in cholangiocyte proinflammatory cytokine expression and proliferation. Our results will yield new mechanistic insights into the normal cholangiocyte functions of recognition and response to microbial pathogens, and may provide a rational basis for the design and implementation of new therapeutic strategies for inflammatory conditions and microbial infection of the biliary tract. PUBLIC HEALTH RELEVANCE: Cholangiocytes are epithelial cells that line intrahepatic bile ducts. These cells are periodically exposed to pathogen-derived toxins (e.g. lipopolysaccharide) or colonized by pathogenic microorganisms (e.g. Cryptosporidium). This proposal focuses on the molecular mechanisms and functional consequences of microbe-induced signaling cascades culminating in a proinflammatory response by cholangiocytes. Our focus will be on microbe recognition-dependent activation of the signaling axis involving the small GTPase, Ras, as well as implications of this activation on the regulated expression of microRNAs, small RNA molecules that regulate gene expression. We propose that this signaling pathway promotes cholangiocyte inflammatory responses, which promotes the attraction of leukocytes to the portal tract.